The water transport properties of the human Na+-coupled glutamate cotransporter (EAAT1) were investigated. The protein was expressed in Xenopus laevis oocytes and electrogenic glutamate transport was recorded by two-electrode voltage clamp, while the concurrent water transport was monitored as oocyte volume changes. Water transport by EAAT1 was bimodal. Water was cotransported along with glutamate and Na+ by a mechanism within the protein. The transporter also sustained passive water transport in response to osmotic challenges. The two modes could be separated and could proceed in parallel. The cotransport modality was characterized in solutions of low Cl- concentration. Addition of glutamate promptly initiated an influx of 436 +/- 55 water molecules per unit charge, irrespective of the clamp potential. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein primarily from the downhill fluxes of Na+ to the uphill fluxes of water. Experiments using the cation-selective ionophore gramicidin showed no unstirred layer effects. Na+ currents in the ionophore did not lead to any significant initial water movements. In the absence of glutamate, EAAT1 contributed a passive water permeability (Lp) of (11.3 +/- 2.0) x 10(-6) cm s(-1) (osmol l(-1))(-1). In the presence of glutamate, Lp was about 50 % higher for both high and low Cl- concentrations. The physiological role of EAAT1 as a molecular water pump is discussed in relation to cellular volume homeostasis in the nervous system.